Journal of Medicinal Chemistry
Article
mixture three times. The organic layer was combined, washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo.
1,3-Dibromo-2-(3-isopropyl-4-methoxyphenoxy)-5-nitrobenzene
(4a). Compound 4a was synthesized from 3-isopropyl-4-methoxyphe-
nol (2a) and 1,3-dibromo-2-iodo-5-nitrobenzene (3a) to give the
22.3. ESI-TOF-MS (−) calcd for C17H16Br2NO3 (M − H)− calcd
439.96 found 439.97. Purity (HPLC−UV): 98% (tR = 10.8 min).
General Procedure E: Suzuki−Miyaura Coupling. To a DME
solution of compound 7 (80 mg, 177 μmol, 1.0 equiv) were added
Pd(PPh3)4 (10 mg, 8.7 μmol, 0.05 equiv), an aqueous solution of
Na2CO3 (400 mg in 1 mL of H2O), and corresponding boronic acid or
boronate ester (212 μmol, 1.2 equiv) in 1 mL of ethanol. This solution
was stirred at 71 °C overnight. After cooling to rt, ethyl acetate and 1
M HClaq were added and extracted 4 times with ethyl acetate. The
organic layer was combined and washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The target compound was purified
by column chromatography and recrystallized from acetone/DCM/
hexane.
1
desired product (1.85 g, 85%) as a yellow powder. H NMR (600
MHz, CDCl3) δ 8.50 (s, 2H, H-7, H-9), 6.81 (d, J = 3.5 Hz, 1H, H-1),
6.71 (d, J = 9.2 Hz, 1H, H-4), 6.43 (dd, J = 8.9, 3.5 Hz, 1H, H-5), 3.79
(s, 3H, CH3O), 3.30−3.28 (m, 1H, isopropyl CH), 1.18 (d, J = 6.9 Hz,
6H, isopropyl (CH3)2). 13C NMR (151 MHz, CDCl3) δ 155.5, 152.9,
150.0, 145.0, 139.3, 128.5, 119.5, 114.4, 111.9, 111.0, 55.9, 27.1, 22.6.
General Procedure B: Deprotection of Methoxy Moiety. To
an ice-cold DCM solution of diphenylether 4, a 1 M boron tribromide
(1.2 equiv) solution in DCM was added dropwise. The reaction was
slowly warmed up to rt and stirred for 1−24 h. To this solution was
slowly added methanol at 0 °C, then warmed to rt. The solution was
washed with saturated NaHCO3 aqueous solution, and the aqueous
layer was extracted with DCM three times. The organic layer was
combined and washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo.
5′-(2,6-Dichloro-4-propionamidophenoxy)-2′-hydroxy[1,1′-bi-
phenyl]-4-carboxamide (8c). Compound 8c was synthesized from
compound 7 and 4-aminocarbonylphenylboronic acid. The product
was obtained as an off-white powder (14 mg, 18%); TLC Rf = 0.4
(DCM/MeOH = 30:1); mp 261.3−262.0 °C; IR (neat) 3339 (broad),
1648, 1592, 1532, 1531, 1462 cm−1; 1H NMR (600 MHz, DMSO-d6)
δ 10.22 (s, 1H, NH), 9.48 (s, 1H, OH), 7.96 (s, 1H, NHH), 7.88−7.83
(m, 2H, biphenyl-H2), 7.82 (s, 2H, H-7, H-9), 7.57−7.55 (m, 2H,
biphenyl-H2), 7.34 (s, 1H, NHH), 6.90 (d, J = 8.8 Hz, 1H, H-4), 6.75
(d, J = 3.1 Hz, 1H, H-1), 6.64 (dd, J = 8.8, 3.2 Hz, 1H, H-5), 2.34 (q, J
= 7.5 Hz, 2H, CH2), 1.09 (t, J = 7.5 Hz, 3H, CH3). 13C NMR (151
MHz, DMSO-d6) δ 172.6, 167.7, 149.7, 149.6, 141.5, 140.6, 137.7,
132.6, 128.7, 128.6, 127.6, 127.2, 119.1, 117.1, 116.0, 114.8, 30.7, 9.4.
4-(2,6-Dibromo-4-nitrophenoxy)-2-isopropylphenol (5a). Com-
pound 5a was synthesized from 4a to give the desired product (1.9
1
g, quant) as a yellow powder; mp 106−107 °C; H NMR (600 MHz,
CDCl3) δ 8.50 (s, 2H, H-7, H-9), 6.79 (d, J = 3.1 Hz, 1H, H-1), 6.65
(d, J = 8.7 Hz, 1H, H-4), 6.39 (dd, J = 8.7, 3.1 Hz, 1H, H-5), 4.54 (s,
1H, OH), 3.18 (septet, J = 6.9 Hz, 1H, isopropyl CH), 1.23 (d, J = 6.9
Hz, 6H, isopropyl (CH3)2). 13C NMR (151 MHz, CDCl3) δ 155.5,
150.2, 148.8, 145.0, 136.7, 128.6, 119.4, 116.0, 114.4, 112.6, 27.5, 22.5.
ESI-TOF-MS (+) calcd for C15H14Br2NO4 (M + H)+ 429.92, found
429.89. Purity (HPLC−UV): 95% (tR = 13.0 min).
+
HRMS (+) calcd for C22H19Cl2N2O4 (M + H) 445.0716. Found
445.0717. Purity (HPLC−UV): 99% (tR = 9.2 min). Anal. C 59.09, H
4.14, N 6.13%, calcd for C22H18Cl2N2O4, C 59.34, H 4.07, N 6.29%.
Cif Preparation. Recombinant Cif-His was prepared as described
previously.5b,26 Stocks of purified proteins were stored at 4 °C until
used. Since esterase and glutathione transferases can give a high
background fluorescence when using cyano(6-methoxynaphthalen-2-
yl)methyl glycidyl carbonate (CMNGC) as Cif substrate, it is
important to evaluate the purity of Cif for the absence of these and
of similar biochemicals. The purified enzyme was tested for
contamination with esterase activity and glutathione transferase
activity as previously described.27
Fluorescent-Based Cif Inhibitory Assay. IC50 values were
determined using a sensitive fluorescent-based assay similar to the
method previously described for other EHs.5b,28 Cyano(6-methoxy-
naphthalen-2-yl)methyl glycidyl carbonate (CMNGC) was used as a
fluorescent reporter substrate. Recombinant Cif (0.6 μM) was
incubated with inhibitors for 5 min in sodium phosphate buffer (20
mM, pH 7.0) containing 50 mM NaCl and 0.1 mg/mL of BSA at 37
°C prior to substrate introduction ([S] = 25 μM).
Activity was measured by determining the appearance of the 6-
methoxy-2-naphthaldehyde with an excitation wavelength of 330 nm
and an emission wavelength of 465 nm for 10 min. Reported IC50
values are the average of triplicates with at least two data points above
and at least two below the IC50. The fluorescent-based assay as
performed here has a standard error between 10% and 20%, suggesting
that differences of 2-fold or greater are significant. Control
experiments without Cif (inhibitor and substrate) were used to
evaluate the intrinsic fluorescence of inhibitors. For compounds
showing IC50 > 50 μM (compounds 1g, 1h, and 1k), solubility under
assay conditions was measured using a method described previously.29
Compounds 1g and 1k showed solubility 50 < S < 100 μM, while
compound 1h showed 25 < S < 50 μM.
Surface Plasmon Resonance. The interactions between Cif and
inhibitors were analyzed by surface plasmon resonance using a
BIAcore T100. The running buffer for immobilization was HBS-P (pH
7.4), which contains 10 mM HEPES, 150 mM NaCl, and 0.05% (v/v)
Tween 20 surfactant. Cif was coupled to the surface of a CM5 sensor
chip using standard amine-coupling chemistry with a 7 min injection of
Cif (15 μg/mL) diluted in 10 mM sodium acetate (pH 4.5). Cif
protein was immobilized at 4000−5000 RU. Remaining activated
groups were blocked with a 7 min injection of 1 M ethanolamine HCl
(pH 8.5). Binding assays were performed at 37 °C at a flow rate of 30
μL/min with a 40 s injection of inhibitors followed by washing with
General Procedure C: Reduction of Nitro Moiety to a
Primary Amine. To a THF solution of compound 5 (1.0 equiv) was
added an aqueous solution of sodium dithionite (3.8 equiv) at rt, then
the solution was heated to 50 °C and stirred 1−24 h. After cooling to
rt, aqueous 1 M HCl was added, and then the solution was neutralized
by a solution of saturated NaHCO3. The organic layer was separated,
and the aqueous layer was extracted with ethyl acetate. The organic
layer was combined and washed with brine, dried over MgSO4, filtered,
and concentrated in vacuo.
4-(4-Amino-2,6-dibromophenoxy)-2-isopropylphenol (6a). Com-
pound 6a was synthesized from 5a to give the desired product (1.38 g,
1
87%) as yellowish crystals; mp 186−187 °C; H NMR (600 MHz,
DMSO-d6) δ 8.93 (s, 1H, OH), 6.87 (s, 2H, H-7, H-9), 6.64 (d, J = 8.7
Hz, 1H, H-4), 6.60 (d, J = 3.1 Hz, 1H, H-1), 6.25 (dd, J = 8.7, 3.1 Hz,
1H, H-5), 5.53 (s, 2H, NH2), 3.14 (septet, J = 6.9 Hz, 1H, isopropyl
CH), 1.10 (d, J = 6.9 Hz, 6H, isopropyl (CH3)2). 13C NMR (151
MHz, DMSO-d6) δ 150.1, 149.1, 148.2, 137.8, 135.3, 118.0, 117.0,
115.2, 112.6, 111.3, 26.5, 22.4. ESI-TOF-MS (+) calcd for
C15H16Br2NO2 (M + H)+ 399.95, found 399.94. Purity (HPLC−
UV): 99% (tR = 10.9 min).
General Procedure D: Amide Bond Formation. To an ice-cold
MTBE solution of aniline 6 (1.0 equiv) was added a solution of
saturated NaHCO3 or 1 M NaOH, followed by slowly adding the
corresponding acid chloride (1.1 equiv). This solution was stirred at rt
for 1−24 h, then 1 M HCl aqueous solution was added. The reaction
mixture was extracted with ethyl acetate. The organic layer was
combined and washed with 1 M HClaq twice, saturated aqueous
NaHCO3 solution 4 times, and brine, dried over MgSO4, filtered, and
concentrated in vacuo. The target compound was purified by column
chromatography and further purified by recrystallization from
acetone/DCM/hexane when possible.
N-(3,5-Dibromo-4-(4-hydroxy-3-isopropylphenoxy)phenyl)-
acetamide (1b). Compound 1b was synthesized from 6a and acetyl
chloride to give the desired product (99 mg, 56%) as a white powder;
1
mp 172−173 °C; H NMR (600 MHz, DMSO-d6) δ 10.24 (s, 1H,
NH), 9.02 (s, 1H, OH), 7.96 (s, 2H, H-7, H-9), 6.67−6.64 (m, 2H, H-
1, H-4), 6.26 (dd, J = 9.4, 2.2 Hz, 1H, H-5), 3.16−3.14 (m, 1H,
isopropyl CH), 2.07 (s, 3H, COCH3), 1.11 (d, J = 7.0 Hz, 6H,
isopropyl (CH3)2). 13C NMR (151 MHz, DMSO-d6) δ 168.8, 149.5,
149.3, 143.8, 138.2, 135.6, 122.7, 117.8, 115.3, 112.8, 111.4, 26.5, 24.0,
G
J. Med. Chem. XXXX, XXX, XXX−XXX